1. Field of the Invention
The present invention concerns improved immunogens containing peptides. More particularly, the invention relates to peptides with an attached hydrophobic tail for adsorption to hepatitis B surface antigen.
2. Background Information
Peptides corresponding to surface regions of different viruses or of other infectious agents may represent potential immunogens with application for vaccination against the respective infectious diseases. However, such peptides frequently require a combination of carriers and adjuvants to become sufficiently immunogenic for consideration as vaccines.
In the case of hepatitis B virus (HBV), the preS1 and preS2 region of the hepatitis B virus envelope protein elicits virus-neutralizing protective antibodies. However, these regions of the hepatitis B virus envelope protein are usually underrepresented in preparations of hepatitis B virus surface antigen (HBsAg) obtained from plasma or from other sources based on recombinant DNA technology. This underrepresentation of preS2 and especially of preS1 sequences is due to a blockade of secretion from eukaryotic cells of HBsAg containing these sequences. Therefore, a combination of synthetic peptides derived from the preS2 and especially from the preS1 sequence with HgsAg particles, consisting predominantly of the S-protein sequence is expected to result in an improved immunogen eliciting protective virus neutralizing antibodies of the broadest possible specificity (A. R. Neurath, S. B. H. Kent, "The PreS Region of Hepadnavirus Envelope Proteins", in K. Maramorosh, F. A. Murphy, A. J. Shatkin, eds., Advances in Virus Research, Vol. 32, Orlando, Fla., Academic Press, (1987)).
The addition to hepatitis B virus surface antigen of polypeptide sequences expected to elicit protective antibodies against hepatitis B (or against other unrelated infectious agents) can theoretically be accomplished by chemically linking synthetic peptides to HBsAg. Such linkage requires chemically active groups on both the peptide and on the surface of HBsAg (for example, SH groups, epsilon-amino groups of lysines, or N-terminal amino groups). HBsAg does not have available free SH groups, since the cysteine residues of HBsAg are all involved in the formation of disulfide bonds within the HBsAg protein. These bonds are absolutely necessary for the antigenicity and immunogenicity of HBsAg particles. The epsilon-amino groups on the surface of HBsAg play important roles in the immunogenicity and antigenicity of HBsAg as well (reviewed by A. R. Neurath and S. B. H. Kent in Immunochemistry of Viruses, "The Basis of Sero-Diagnosis and Vaccines", pp. 324-357, eds. M. H. V. Van Regenmortel and A. R. Neurath, Elsevier, Amsterdam, (1985)). For this reason, the chemical linkage of sufficient quantities of synthetic peptides to HBsAg particles cannot be accomplished without resulting in a deleterious effect on the antigenicity and immunogenicity of S-protein. For this reason, there has been a great need to find methods which would allow the attachment of synthetic peptides to HBsAg, not involving a chemical linkage, but strong enough to allow the HBsAg protein to function as a carrier for the synthetic peptide and for the only approved adjuvant alum to function as an adjuvant for HBsAg and the synthetic peptides of choice
HBsAg has on its surface exposed hydrophobic regions which interact strongly with hydrophobic (long aminoalkyl, alkyl or aromatic chains) residues exposed on the surface of various adsorbents Such hydrophobic interaction is the basis of the method to purify HBsAg from serum and from other biological fluids (U.S. Pat. No. 3,976,767, issued Aug. 24, 1976).
______________________________________ DEFINITIONS ______________________________________ Amino Acid Code Words D Asp aspartic acid N Asn asparagine T Thr threonine S Ser serine E Glu glutamic acid Q Gln glutamine P Pro proline G Gly glycine A Ala alanine C Cys cysteine V Val valine M Met methionine I Ile isoleucine L Leu leucine Y Tyr tyrosine F Phe phenylalanine W Trp tryptophane K Lys lysine H His histidine R Arg arginine HBV hepatitis B virus HBsAg hepatitis B surface antigen DNA deoxyribonucleic acid ______________________________________